Project Summary/Abstract Well powered genome-wide association studies (GWAS) for celiac disease and Crohn's disease have identified numerous non-HLA susceptibility loci. Genetic fine-mapping and expression quantitative trait loci (eQTL) can narrow in on which genes probably account for the GWAS signals, but even after these approaches the locations of the causal nucleotide changes often remain unknown. This gap in understanding is a roadblock to targeted prevention and therapy. As developed in our prior work, mapping haplotype-dependent allele-specific CpG methylation (hap-ASM) and methylation quantitative trait loci (mQTLs) in cells relevant to a given disease, and then overlapping these epigenetic maps with GWAS data, can help to hone in on the DNA regulatory sequences that causally underlie the GWAS signals. Our hypothesis is that this combined genetic-epigenetic mapping strategy, followed by functional assays, will be able to identify regulatory DNA sequences that contribute to celiac disease and Crohn's disease susceptibility and pathogenesis. First, we will carry out Methyl-Seq of CD4+ and CD8+ T cells and peripheral blood monocytes, to map hap-ASM genome-wide. These data will pinpoint hap- ASM differentially methylated regions (DMRs) in the same haplotype blocks as GWAS peaks for celiac and Crohn's. Since hap-ASM often reflects allele-specific transcription factor binding site (TFBS) or insulator occupancies, we will cross-validate our findings using an independent method, Assay for Transposase- Accessible Chromatin Sequencing (ATAC-Seq). In our second aim, we will rank and prioritize the hap-ASM DMRs based on the strength of the allelic asymmetries, ATAC-Seq overlaps, and linkage disequilibrium (LD) with GWAS peak SNPs, and perform high-throughput targeted bisulfite sequencing to fine-map the top-ranked DMRs, both in the blood-derived cells and in mucosal T cells from celiac and Crohn's patients. In our third aim, to definitively test the functional roles of specific TFBS in the DMRs, we will use CRISPR to delete these sequences in Jurkat cells and in normal T cells. We will score effects of the deletions on local methylation patterns and mRNA levels of each of the nearby genes. These data will clarify our fundamental understanding of susceptibility and pathogenesis of celiac disease and Crohn's disease.